In the midst of all this hoopla about reliability, repeatability, the replication crisis and what not the Editorial Board of the Journal of Neuroscience has launched an effort to recommend best practices. The first one was about electrophysiology. To give you a flavor:

There is a long tradition in neurophysiology of using the number of neurons recorded as the sample size (“n”) in statistical calculations. In many cases, the sample of recorded neurons comes from a small number of animals, yet many statistical analyses make the explicit assumption that a sample constitutes independent observations. When multiple neurons are recorded from a single animal, however, either sequentially with a single electrode or simultaneously with multiple electrodes, each neuron's activity may not, in fact, be independent of the others. Thus, it is important for researchers to account for variability across subjects in data analyses.

I emphasize the "long tradition" part because clearly the Editorial Board does not just mean this effort to nibble around the edges. It is going straight at some long used practices that they think need to change.

There was a long and very good twitter thread from someone which dealt in part with unreliability relating to when one chooses to conduct behavioral tasks in rodents with respect to their daily light cycle. As a reminder, rodents are nocturnal and are most active (aka "awake") in the dark. Humans, as a reminder, are not. So, as you might imagine, there is a lot of rodent research (including behavioral research) that fails to grasp this difference and simply runs the rats in their light cycle. Also known as their inactive part of the day. Aka. "asleep".

Which reminds me to tell @MarinaP63 that her upcoming “recommendations” on behavioral experiments better insist on banning light cycle “behavior” for rodents.

There are just too many ludicrous things being said to characterize them all. But, one species of argument is "it doesn't matter [for my endpoint]". The last part is implied. But early in this thread I posted a link to my prior post which discusses two of my favorite papers on this topic. Scheving et al, 1968 showed a four fold difference in mortality rate after a single dose of amphetamine depending on when it was administered. Roberts and colleagues showed that cocaine self-administration changes all across the day in a very nice circadian pattern. I also noted a paper I had discussed very indirectly in a post on contradicting your own stuff. Halberstadt and colleagues (2012) played around with some variables in a very old model from the Geyer lab and found that time of day interacted with other factors to change results in a rat locomotor assay. I mean c'mon, how many thousands of papers use locomotor assays to asssess psychomotor stimulant drugs?

There's some downshifting and muttering in the tweet discussion about "well if it doesn't matter who cares" but nobody has started posting published papers showing where light cycle doesn't matter for their assays (as a main factor or as an interaction). Yet. I'm sure it is just an oversight. Interestingly the tone of this seems to be arguing that it is ridiculous to expect people to do their rat assays in reverse light unless it is proven (I guess by someone else?) that it changes results.

This, my friends, is very much front and center in the "reproducibility crisis" that isn't. Let us return to the above comment at J Neuro about "long traditions". Do you know how hard it is to fight long traditions in scientific subareas? Sure you do. Trying to get funded, or publish resulting studies, that deal with the seemingly minor choices that have been made for a long time is very difficult. Boring and incremental. Some of these things will come out to be negative, i.e., it won't matter what light cycle is used. Good luck publishing those! It's no coincidence that the aforementioned Halberstadt paper is published in a very modest journal. So we end up with a somewhat random assortment of some people doing their work in the animals' inactive period and some in the active period. Rarely is there a direct comparison (i.e., within lab). So who knows what contribution that is....until you try to replicate it yourself. Wasting time and money and adding potential interactions.....very frustrating.

So yes, we would like to know it all, kind of like we'd like to know everything in male and female animals. But we don't. The people getting all angsty over their choice to run rodents in the light next tried the ploy to back and fill with "can't we all get along" type of approach that harmonizes with this sentiment. They aren't wrong, exactly. But let us return to the J Neuro Editorial effort on best practices. There IS a best option here, if we are not going to do it all. There's a slope in your choice of default versus checking the other. And for behavioral experiments that are not explicitly looking at sleepy rats or mice, the best option is running in their active cycle.

There is lots of fun ridiculousness in the thread. I particularly enjoyed the argument that because rats might be exposed briefly to light in the course of trying to do reverse-cycle experiments, we should just default to light cycle running. Right? Like if you walk from the light into a darkened room you suddenly fall asleep? Hell no. And if you are awakened by a light in your face in the middle of the night you are suddenly as awake as if it were broad noon? HAHAHHAHAA! I love it.

Enjoy the threads. Click on those tweeter links above and read the arguments.

Blessings on you for fighting this good fight. We circadian biologists are universally horrified by the fast and loose way that people play with random circadian times for experimentation. For most behaviors, animals should be tested during their active phase. Preferably within a short time window, because many behaviors change even within the active period. Behaviors like aggression, courtship, attention, learning, memory, fear, addiction all change. Because physiological factors ALSO change around the circadian day, including immune response, vasodilation, body temperature, blood pressure, uptake of xenobiotics by many tissues, etc.

Organisms on planet earth evolved in a light cycle that affects many environmental factors: temperature, food availability, predator prevalence, mate availability and more. So it makes absolute sense that creatures from algae to plants to flies to mice to humans have behavior and physiology gated by circadian rhythms.

And it's also worth remembering that the microbiota have their own circadian rhythms in addition to being influenced by the host rhythms. Given the growing realization of the effect of the microbiota on other variables of interest, there's multiple reasons to take time of day into account.

Yes, we should all switch to gerbils, which aren't nocturnal. Of course there's the slight problem that our knowledge of gerbil genetics is no where close to the level of mouse/rat genetics and there aren't the knockout strains needed to do any current science. This is the problem with model organisms in hindsight -- they are rarely the ones one would pick with the knowledge we have today. E. coli, with its many redundant pathways and its ability to live both aerobically and anaerobically isn't the ideal bacterial model either. And yet as with mice/rats our vast knowledge and tools in E. coli make it difficult to switch.

I vote for guinea pigs, because they have periods of activity during both light and darkness, much like modern humans in light polluted environments.
As a bonus, they are edible, and if we could at least use the naive control animals as food it would be morally good.

Ironically they *were* an early subject of animal research -- hence the jocular term "guinea pig" for human test subjects. But their size and longer gestation period caused them to be mostly replaced post WWII with mice and rats.

@JB. I used guinea pigs for my work on Legionnaire's disease in the 1980s as they were the only small mammal susceptible to lethal infection following inhalation of low concentration aerosols of the bug. Of course back then mice, rats, guinea pigs were all on an essentially even playing field (i.e. we knew almost bugger all how their immune systems functioned). Nevertheless, it is still almost easier to get blood out of a stone than a guinea pig save for the penile vein or sequential cardiac punctures. The latter brings high mortalities. We used predominantly females and could barely get 100 ul blood from them by nicking the vasculature between their toes. This still makes them an "only if we have to" species for lab animal work.